The free, or non-bound, fraction of many drugs and hormones in blood or serum is of great interest to clinical and pharmaceutical chemists because this fraction is believed to represent the active form of many such agents. As a result, it is the free fraction of these drugs and hormones that should ideally be measured as a tool for patient diagnosis and treatment. However, there is currently no general and fast approach for measuring the free fractions of drugs and hormones in clinical samples. The overall goals of this proposal are 1) to obtain a better understanding of drug and hormone interactions with their binding agents in blood or serum and 2) to use this information to develop fast, reliable free drug and hormone assays. The underlying hypothesis of this project is that a new class of improved free drug and hormone assays can be created based on the techniques of ultrafast affinity extraction and chromatographic immunoassays, as recently demonstrated for compounds such as warfarin, thyroxine, phenytoin and carbamazepine. Future studies will build on these previous efforts by considering new analytes and approaches that can be employed in such work. Particular emphasis will be given to high-throughput methods and techniques that can be adapted for the detection of a broad range of analytes. Advances to be explored in the field of binding studies will include the development of rapid affinity methods for examining both the extent and rate of drug or hormone interactions with serum agents, and the use of affinity microcolumns as tools in personalized medicine to study changes in drug interactions with variants of serum proteins. Improved tools to be created for free drug and hormone measurements will include ultra-high capacity affinity supports for use in fast drug or hormone extractions, chromatographic immunoassays with on-column detection, multi-dimensional systems for free drug/hormone assays, the use of serum protein columns to carry out free drug/hormone assays, and improved analytical platforms for improving the flexibility and sample throughput when using chromatographic immunoassays with near infrared fluorescent labels. An immediate impact of this work will be a better understanding of how drugs and hormones interact with serum binding agents and are transported in the circulatory system. In addition, the new techniques that will be created for free drug/hormone measurements should greatly improve the ability of clinicians and pharmaceutical chemists to study and examine the biological effects of drugs and hormones in the body. This capability should lead to better protocols in clinical testing and in personalized medicine for the treatment and diagnosis of patients suffering from a variety of disorders. The various affinity-based approaches developed in this project for examining solute-ligand binding, rapidly extracting analytes from biological samples, and measuring trace levels of such analytes should also be valuable in many areas of biochemical research, including proteomics, metabolomics and the characterization or screening of drug candidates.